Name: the double-h maze: a robust behavioral test for learning and memory in rodents
Uploaded: 2015-07-08T13:00:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents at JoVE.com

This work was supported by the University of Strasbourg and Neurex&#8212;Neuroscience Upper Rhine Network (post-doc fellowship to RP) and by BrainLinks-BrainTools Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086). We thank Nadja Martini for expert technical assistance.

1. General Considerations
This protocol is approved by the Animal Care and Use Committee of University Hospital Freiburg (same for Strasbourg). Visual acuity is necessary for performance in tests of spatial learning. Rodents with impaired visual systems are thus not suitable. Also, lighting must be sufficient in order for the rats to see the different cues located on the surrounding walls. It is useful to utilize basic-shaped (square, circle, triangle) but well-contrasted cues (e.g., black-painted cues on a white-painted background). Likewise, severe motoric deficits are exclusion criteria because swimming is required for this test and drowning may occur. Finally, hyper-anxious rodents can display a strongly biased search behavior, which impacts on performance.
2. Apparatus Set Up
<ol>
	<li>Construct a double-H maze consisting of a 160 cm central alleyway, which is intersected at both ends and in the center by three 160 cm parallel run arms (see Figure 1). Ensure that the central alleyway and its perpendicular arms are 20 cm wide, and are surrounded by 35 cm high transparent Plexiglas walls. Thickness of the Plexiglas is 6 mm for all parts.</li>
	<li>Secure the walls in place using glue and screws, and waterproof the maze using silicone joints at all internal angles. These joints can be replaced easily should they lose their waterproofness. Place a drain outlet at the extremity of one of the corner arms (or in the middle of the maze) for emptying.</li>
	<li>Place the maze on an 80 cm high table, with adequate space around it for a) walking around the maze, and b) placement of well-contrasted cues. Designate each arm in the maze by its position, i.e., northwest (NW), north (N), northeast (NE), southwest (SW), south (S), and southeast (SE).</li>
	<li>Place a ceiling-mounted camera above the maze for post-test analysis of animal behavior, using either manual or automatic (video-tracking) methods (see &#167;5).</li>
</ol>
3. General Comments
<ol>
	<li>Prior to use, fill the maze with water to an approximate height of 18 cm (200 L). This is high enough to prevent rats from touching the bottom of the maze with their feet, but shallow enough to prevent escape.</li>
	<li>After pre-training, render the water opaque by mixing 250 g skim milk powder. Change the water on a daily basis to prevent the milk-water from becoming rancid. Maintain the water temperature between 21 - 23 &#176;C to provide incentive for the rats to seek the escape platform. 
	NOTE: As rats become familiar with the position of the platform during pre-training, its position is moved to a different arm during training.</li>
	<li>Prior to use, immerse a 17 cm high, 10 cm diameter platform at the extremity of one of the corner arms (NE, NW, SE or SW). Ensure the height to be submerged is 1 cm below the surface of the water surface. Train the rats to reach the target platform using either an allocentric or egocentric learning strategy, which is dependent on the type of paradigm used (see &#167;4).</li>
</ol>
4. Basic Training Protocols
NOTE: Rats are typically provided with an initial day of pre-training, which allows them to become familiar with the maze.
<ol>
	<li>For pre-training release the rat from the extremity of one of the center arms (e.g., S arm) and place the goal platform at the extremity of one of the corner arms (e.g., NE), then give the rats 4 consecutive 60 sec trials in which to reach the target platform.</li>
	<li>Upon reaching the escape platform, allow the rat to wait there for 15 sec, so that they can rest and observe their surroundings. Regardless of the start position, always block the opposite arm with a transparent guillotine door, which prevents entry. 
	NOTE: During pre-training, leave the water transparent, and adjust the platform height such that it protrudes 1 cm above the water&#8217;s surface, thus making it visible to the rat.</li>
	<li>Perform daily training session consisting of up to 4 consecutive trials, separated by a 10 sec gap at least (discrete training, i.e., with intervals of several minutes between trials, is an alternative).</li>
	<li>For training, relocate the platform from its pre-training position to the chosen arm (e.g., NW), and submerge it at its extremity 1 cm below the water surface. Now render the water opaque by addition of milk power and perform the following training (section 4).</li>
	<li>For rats that do not reach the target platform within 60 sec, return them to the starting position, and gently guide them to the platform by the experimenter.</li>
	<li>Measure several variables during the training and probe sessions, such as, distance swam, latency to goal arm / platform, time spent in each arm, as well as the number of initial/repetitive errors (see Figure 2). Keep in mind that latencies may be influenced by motor difficulties. Should the case arise, distance and errors appear as more reliable variables regarding cognitive performance.</li>
	<li>Allocentric Strategy Training:
	<ol>
		<li>Day&#160;1 - Pre-Training:
		<ol>
			<li>Do not add milk powder for this step. Place escape platform protruding 1 cm above the water&#8217;s surface in a fixed location. Train rats in 4 consecutive trials to reach the platform.</li>
		</ol>
		</li>
		<li>Days 2 - 5 &#8211; Training:
		<ol>
			<li>Add 250 g of skim milk powder to the water to render it opaque. Move the platform to a different arm (e.g., from NE to NW) and add water so that the platform is 1 cm below the water surface. Release rats from either the N or S arm in unpredictable sequences for each session, such that both arms are used twice as trial start in a single session (4 trials/day, e.g., SNNS, NSNS; see Figure 3).</li>
		</ol>
		</li>
		<li>Day 6 &#8211; Probe Session:
		<ol>
			<li>Remove platform for the probe trial. Release rats from a different arm to those used during training (e.g., SW), and allow them to swim for 60 sec. See Figure 4 for representative swim tracks. Analysis of the time spent in the target arm (former location of the platform) gives an indication about whether rats use a spatial strategy, another type of strategy, a sequential combination of different strategies (see below), or a disorganized search pattern.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Egocentric Strategy Training:
	<ol>
		<li>Day 1 &#8211; Pre-Training:
		<ol>
			<li>This is the same as first step in the allocentric strategy training (step 4.1). Do not add milk powder for this step. Place escape platform protruding 1 cm above the water&#8217;s surface in a fixed location. Train rats in 4 consecutive trials to reach the platform.</li>
		</ol>
		</li>
		<li>Days 2 - 5 &#8211; Training:
		<ol>
			<li>Add 250 g of skim milk powder to the water to render it opaque. Move the platform to a different arm and add water so that the platform is 1 cm below the water surface. Release rats from the same start arm (S or N; see Figure 5) for every trial (4 trials/day).</li>
		</ol>
		</li>
		<li>Day 6 &#8211; Probe Session:
		<ol>
			<li>Remove platform for the probe trial. Rats are released from an arm different from where they were released during the training. Allow the rats to swim for 60 sec. Block the opposite arm with a transparent guillotine door. 
			NOTE: Modify the above training strategies for according to the requirements of the particular experiment, e.g., Testing Drug Effects etc.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>
	Dry the rat off with absorbent towels after each session in the water.
	</li>
</ol>
5. Analysis
<ol>
	<li>Perform measurements of latencies, initial and repetitive errors, first choices and response type manually by recording these variables from the videos taken from the overhead camera.</li>
	<li>Alternatively if available, utilize commercially available video-tracking software and configured to record these variables automatically.</li>
	<li>Statistical Analysis: 
	NOTE: The specific implementation of statistical analysis depends on the study, which is taking place.
	<ol>
		<li>Perform a one, two or three-way ANOVAs regarding the initial/repetitive errors, latency to goal arm/platform, and time spent in the target arm; with factors that include the test day, and treatment group(s).</li>
		<li>Where necessary, follow these ANOVAs using post-hoc Newman-Keuls multiple comparisons tests. To compare performance, use a reference value (e.g., the time spent in the target arm during a probe trial vs. the chance level), and perform t-test.</li>
		<li>If required, use non parametric statistics in addition (e.g., &#967;&#178;, see below) or instead when conditions for parametric ones are not fulfilled.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Izquierdo, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+connection+between+the+hippocampal+and+the+striatal+memory+systems+of+the+brain:+a+review+of+recent+findings.">The connection between the hippocampal and the striatal memory systems of the brain: a review of recent findings.</a> Neurotoxicity Research. 10 (2), 113-121 (2006).</li><li>Albouy, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+the+hippocampus+and+striatum+are+involved+in+consolidation+of+motor+sequence+memory.">Both the hippocampus and striatum are involved in consolidation of motor sequence memory.</a> Neuron. 58 (2), 261-272 (2008).</li><li>Albouy, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Hippocampal+and+Striatal+Systems+Predicts+Subsequent+Consolidation+of+Motor+Sequence+Memory.">Interaction between Hippocampal and Striatal Systems Predicts Subsequent Consolidation of Motor Sequence Memory.</a> Plos One. 8 (3), (2013).</li><li>Chang, Q., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-hippocampal+lidocaine+injections+impair+acquisition+of+a+place+task+and+facilitate+acquisition+of+a+response+task+in+rats.">Intra-hippocampal lidocaine injections impair acquisition of a place task and facilitate acquisition of a response task in rats.</a> Behav Brain Res. 144 (1-2), 19-24 (2003).</li><li>McDonald, R. J., Hong, N. S., Devan, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+understanding+mammalian+cognition+and+memory-based+behaviours:+an+interactive+learning+and+memory+systems+approach.">The challenges of understanding mammalian cognition and memory-based behaviours: an interactive learning and memory systems approach.</a> Neurosci Biobehav Rev. 28 (7), 719-745 (2004).</li><li>Packard, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anxiety+cognition,+and+habit:+a+multiple+memory+systems+perspective.">Anxiety cognition, and habit: a multiple memory systems perspective.</a> Brain Res. 1293, 121-128 (2009).</li><li>Packard, M. G., McGaugh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+hippocampus+or+caudate+nucleus+with+lidocaine+differentially+affects+expression+of+place+and+response+learning.">Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning.</a> Neurobiol Learn Mem. 65 (1), 65-72 (1996).</li><li>Lawrence, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+and+mnemonic+functions+in+early+Huntington's+disease.">Executive and mnemonic functions in early Huntington's disease.</a> Brain. 119 (Pt 5), 1633-1645 (1996).</li><li>Lawrence, A. D., Watkins, L. H., Sahakian, B. J., Hodges, J. R., Robbins, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+object+and+visuospatial+cognition+in+Huntington's+disease:+implications+for+information+processing+in+corticostriatal+circuits.">Visual object and visuospatial cognition in Huntington's disease: implications for information processing in corticostriatal circuits.</a> Brain. 123 (Pt 7), 1349-1364 (2000).</li><li>Walker, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease.">Huntington's disease.</a> Lancet. 369 (9557), 218-228 (2007).</li><li>Cushman, L. A., Stein, K., Duffy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+navigational+deficits+in+cognitive+aging+and+Alzheimer+disease+using+virtual+reality).">Detecting navigational deficits in cognitive aging and Alzheimer disease using virtual reality).</a> Neurology. 71 (12), 888-895 (2008).</li><li>Liu, L., Gauthier, L., Gauthier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+disorientation+in+persons+with+early+senile+dementia+of+the+Alzheimer+type.">Spatial disorientation in persons with early senile dementia of the Alzheimer type.</a> A`m J Occup Ther. 45 (1), 67-74 (1991).</li><li>Mapstone, M., Steffenella, T. M., Duffy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visuospatial+variant+of+mild+cognitive+impairment:+getting+lost+between+aging+and+AD.">A visuospatial variant of mild cognitive impairment: getting lost between aging and AD.</a> Neurology. 60 (5), 802-808 (2003).</li><li>Vliet, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropsychological+profiles+of+mild+Alzheimer's+disease+and+questionable+dementia+as+compared+to+age-related+cognitive+decline.">The neuropsychological profiles of mild Alzheimer's disease and questionable dementia as compared to age-related cognitive decline.</a> J Int Neuropsychol Soc. 9 (5), 720-732 (2003).</li><li>Hanagasi, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+impairment+in+amyotrophic+lateral+sclerosis:+evidence+from+neuropsychological+investigation+and+event-related+potentials.">Cognitive impairment in amyotrophic lateral sclerosis: evidence from neuropsychological investigation and event-related potentials.</a> Brain Res Cogn Brain Res. 14 (2), 234-244 (2002).</li><li>Robbins, T. W., Murphy, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+pharmacology:+40++years+of+progress,+with+a+focus+on+glutamate+receptors+and+cognition.">Behavioural pharmacology: 40+ years of progress, with a focus on glutamate receptors and cognition.</a> Trends Pharmacol Sci. 27 (3), 141-148 (2006).</li><li>Paul, C. M., Magda, G., Abel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+memory:+Theoretical+basis+and+comparative+review+on+experimental+methods+in+rodents.">Spatial memory: Theoretical basis and comparative review on experimental methods in rodents.</a> Behav Brain Res. 203 (2), 151-164 (2009).</li><li>Hodges, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+procedures:+the+radial-arm+and+water+maze+compared.">Maze procedures: the radial-arm and water maze compared.</a> Brain Res Cogn Brain Res. 3 (3-4), 167-181 (1996).</li><li>Kesner, R. P., Farnsworth, G., Kametani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+parietal+cortex+and+hippocampus+in+representing+spatial+information.">Role of parietal cortex and hippocampus in representing spatial information.</a> Cereb Cortex. 1 (5), 367-373 (1991).</li><li>Barnes, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+deficits+associated+with+senescence:+a+neurophysiological+and+behavioral+study+in+the+rat.">Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat.</a> J Comp Physiol Psychol. 93 (1), 74-104 (1979).</li><li>Whishaw, I. Q., Cassel, J. C., Jarrad, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats+with+fimbria-fornix+lesions+display+a+place+response+in+a+swimming+pool:+a+dissociation+between+getting+there+and+knowing+where.">Rats with fimbria-fornix lesions display a place response in a swimming pool: a dissociation between getting there and knowing where.</a> J Neurosci. 15 (8), 5779-5788 (1995).</li><li>Staay, F. J., Raaijmakers, W. G., Lammers, A. J., Tonnaer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+fimbria+lesions+impair+acquisition+of+working+and+reference+memory+of+rats+in+a+complex+spatial+discrimination+task.">Selective fimbria lesions impair acquisition of working and reference memory of rats in a complex spatial discrimination task.</a> Behav Brain Res. 32 (2), 151-161 (1989).</li><li>Olton, R. S., Samuelson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remembrance+of+places+past:+Spatial+memory+in+rats.">Remembrance of places past: Spatial memory in rats.</a> J Exp Psych. 2 (2), 97-116 (1976).</li><li>Pol-Bodetto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+double-H+maze+test,+a+novel,+simple,+water-escape+memory+task:+acquisition,+recall+of+recent+and+remote+memory,+and+effects+of+systemic+muscarinic+or+NMDA+receptor+blockade+during+training.">The double-H maze test, a novel, simple, water-escape memory task: acquisition, recall of recent and remote memory, and effects of systemic muscarinic or NMDA receptor blockade during training.</a> Behav Brain Res. 218 (1), 138-151 (2011).</li><li>Cassel, R., Kelche, C., Lecourtier, L., Cassel, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+match/mismatch+of+visuo-spatial+cues+between+acquisition+and+retrieval+contexts+influences+the+expression+of+response+vs.+place+memory+in+rats.">The match/mismatch of visuo-spatial cues between acquisition and retrieval contexts influences the expression of response vs. place memory in rats.</a> Behavioural Brain Research. 230 (2), 333-342 (2012).</li><li>Schumacher, A., de Vasconcelos, A. P., Lecourtier, L., Moser, A., Cassel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+high+frequency+stimulation+in+the+dorsal+striatum:+Effects+on+response+learning+and+on+GABA+levels+in+rats.">Electrical high frequency stimulation in the dorsal striatum: Effects on response learning and on GABA levels in rats.</a> Behavioural Brain Research. 222 (2), 368-374 (2011).</li><li>Lecourtier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+neurobehavioral+development+and+dramatic+impairments+of+procedural-like+memory+following+neonatal+ventral+hippocampal+lesion+in+rats.">Intact neurobehavioral development and dramatic impairments of procedural-like memory following neonatal ventral hippocampal lesion in rats.</a> Neuroscience. 207, 110-123 (2012).</li><li>Kirch, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+deficits+in+declarative+and+procedural+memory+dependent+behavioral+function+in+a+transgenic+rat+model+of+Huntington's+disease.">Early deficits in declarative and procedural memory dependent behavioral function in a transgenic rat model of Huntington's disease.</a> Behav Brain Res. 239, 15-26 (2013).</li><li>Cholvin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventral+midline+thalamus+contributes+to+strategy+shifting+in+a+memory+task+requiring+both+prefrontal+cortical+and+hippocampal+functions.">The ventral midline thalamus contributes to strategy shifting in a memory task requiring both prefrontal cortical and hippocampal functions.</a> J Neurosci. 33 (20), 8772-8783 (2013).</li><li>Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Brain Res Brain Res Rev. 36 (1), 60-90 (2001).</li></ol>

The authors have nothing to disclose.

Comments on Study Design and Analysis
Since its conception, the double-H maze has been utilized in a number of behavioral experiments in rats, which collectively were designed to study egocentric and/or allocentric responses in rats under normal24,25 and altered26-29 brain states. The latter studies include striatal deep-brain stimulation (DBS)26, animal models of neurological disorders27,28, as well as bilateral deactivations of various cortico-hippoca...

In animals, learning is principally mediated by the hippocampal- and striatal-based memory systems1,2, which play central roles regarding place- and procedural-memory, respectively. The relationship between these two systems is complex, and they are known to interact with each other in cooperative or competitive manners1,3. In addition, studies have shown that the influence of either of these memory systems on animal behavior can increase following the absence or damage of the other system4-7. Both of these systems are connected to the prefrontal cortex via the thalamus.
Numerous neurological disorders and neurodegenerative diseases can affect spatial cognition in humans, which rely on the interplay between procedural and declarative memory systems. Examples include Parkinson&#8217;s disease (PD), Huntington&#8217;s disease (HD)8-10, Alzheimer&#8217;s disease (AD)11-14, as well as amyotrophic lateral sclerosis (ALS)15. Animal models, which are relevant to these disorders can be induced through various drug treatments which block certain receptors16, as well as through targeted lesions. When such animals are used with spatial memory tasks, a valuable insight can be gained into the underlying mechanisms related to these disorders, as well as to various treatment options.
There are many different types of spatial memory tasks in rodents, which collectively are designed to assess specific aspects of learning and memory, as well as the effects of potential treatments for various disorders17,18. These tasks can be distinguished by the number of goals and pathways, the degree of behavioral flexibility in solving the task, the memory duration or delay, as well as the choice of strategy used in solving the task. A good performance may be acquired based on external cues or landmarks which are used to orientate the animal towards the goal (an allocentric or place strategy). Alternatively, a rodent may develop a strategy which is based on bodily direction and cues with regards to the direction to move in (an egocentric or procedural strategy), e.g., if a rat knows that the goal is one left turn followed by one right turn, then there is little need for an allocentric or place strategy. Maze tasks often differ based on the degree of flexibility offered to the rodent in solving them. For instance, in the Morris Water Maze, a dry version of the latter (e.g., 19) or the Barnes maze (e.g., 20), there are potentially infinite routes the rat can take to reach the goal. In the Morris Water Maze, for example, the location of the goal may be learned based on external landmarks or cues (allocentric strategy), or by simply swimming in circles towards the center until the platform is found (egocentric strategy)21. Certain tasks have multiple goals and a high degree of flexibility, such as the cone-field task22 or Olton&#8217;s radial maze23. At the other end of the scale are tasks, which offer limited flexibility in reaching the goal, e.g., the Stone maze, or the alternating version of the T-maze. These tasks provide only one correct way of reaching the goal and facilitate the emergence of cognitive routines that are principally governed by the striatal-based procedural memory system.
The double-H maze is a novel spatial memory testing device, which was designed to allow the experimenter to direct the type of strategy that is learned by rodents in solving the task24. Consisting of three parallel run arms intersected by a perpendicular central alleyway, the double-H maze is a water-escape task in which rodents learn to reach an escape platform that is immersed in one of the maze locations. During training, a procedural strategy can be developed by maintaining the same start and goal locations throughout. Alternatively, an allocentric strategy may be developed by alternating the starting location in a random order, thus requiring the rat to learn the location of the hidden platform based on environmental cues as it has to do in a water maze. This overcomes an obstacle present in many different maze tasks, in which the experimenter otherwise has little control over the type of strategy that rodents utilize. This is important when considering that the effects of certain cognition-enhancing drug candidates rely on the hippocampal-based place-memory system, thus the emergence of cognitive routines or procedures may confound the interpretation of the behavioral observations when animals, for example switch from allocentric to procedural memory during the course of training. Similarly, it may be desirable to assess the effects of drugs and treatments on procedural memory, without the influence of allocentric place-based memory. Finally, this device can be utilized to study the cooperative or competitive interactions between these memory systems, and the conditions under which rodents may switch from one system to another.

<table><tbody><tr><td>Rats or Mice</td><td>Charles River</td><td></td><td></td></tr><tr><td>Towels for drying</td><td>University Hospital</td><td></td><td>1 / animal</td></tr><tr><td>Water</td><td></td><td></td><td>~200 L / day</td></tr><tr><td>Skim milk powder</td><td>Grocery store</td><td></td><td>250 g / 200 L water</td></tr><tr><td>Garden Hose</td><td>Hardware store</td><td></td><td></td></tr><tr><td>Drying rack for towels</td><td>Hardware store</td><td></td><td></td></tr><tr><td>Kinect camera</td><td>Kinect</td><td></td><td></td></tr><tr><td>PC computer</td><td>any</td><td></td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Double H Maze, (plexiglass)&lt;/strong&gt; (Custom-Built)</td></tr><tr><td>External lateral walls, 1600 &amp;times; 350 &amp;times; 6 mm</td><td></td><td></td><td>2</td></tr><tr><td>Internal lateral walls, 706 &amp;times; 350 &amp;times; 6 mm</td><td></td><td></td><td>8</td></tr><tr><td>Central corridor panels, 500 &amp;times; 350 &amp;times; 6 mm</td><td></td><td></td><td>4</td></tr><tr><td>Arm extremities, 188 &amp;times; 350 &amp;times; 6 mm</td><td></td><td></td><td>6</td></tr><tr><td>Guillotine doors, 187 &amp;times; 350 &amp;times; 6 mm</td><td></td><td></td><td>3</td></tr><tr><td>Extremity covers, 200 &amp;times; 250 mm</td><td></td><td></td><td>6</td></tr><tr><td>Crossbars, 200 &amp;times; 40 mm</td><td></td><td></td><td>6</td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;Double-H Maze Platform (to be ballasted)&lt;/strong&gt; (Custom-Built)</td></tr><tr><td>Metal platform, 100 mm diameter &amp;times; 150 mm</td><td></td><td></td><td>2</td></tr><tr><td>Platform cover, 100 mm diameter &amp;times; 6 mm</td><td></td><td></td><td>2</td></tr></tbody></table>

the double-h maze: a robust behavioral test for learning and memory in rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

Egocentric Learning Strategy
A study was carried out to determine whether the chosen memory strategy in rats changes based on alterations of their perspective of external environmental cues, following an egocentric-learning paradigm25. Rats were trained over 4 days (4 trials/day) to reach a goal arm located at NE, and were subsequently tested on the fifth day using a misleading probe trial, in which the start arm was either moved 60 cm to the left (i.e., NE start for animals r...

Watch this Scientific Journal Video about The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents at JoVE.com

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

the-double-h-maze-robust-behavioral-test-for-learning-memory

Universidad San Sebastian

Research

JoVE Journal

Behavior

12.5K Views.  University Hospital Freiburg. The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Video: The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Neuroscience

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

Spatial Memory Testing Using Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
Here, the neurobiology behind spatial memory is briefly reviewed and some general principles behind maze tests are discussed. Then, the video explains generalized protocols on how to conduct trials with specific types of mazes. Lastly, it will explain how behavioral researchers are using these tools to conduct some specific experiments.

Spatial Memory Tests Using Morris Water, Radial Water &amp; Barnes Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this ...

Education

JoVE Science Education

Psychology

Behavioral Science

The Morris Water Maze

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, rodents must swim to a platform to escape from a pool of water. The platform is then hidden under the water&#8217;s surface, so that the animal is required to remember it&#8217;s location in order to escape.This simple yet powerful maze design can be used to assay cognitive function, study animal models of neurodegenerative disease, and test potential drug therapies.
This video provides an introduction to the Morris water maze and the principles surrounding its use, including a discussion of the different types of memory tested in the maze, important points to consider when designing and conducting this experiment, and the procedures for setup and running of the test. Several applications of the maze are examined, such as investigating how radiation treatment may lead to memory impairment. Finally, other types of water mazes, such as the 8-arm radial maze, are introduced to show how this paradigm can be adapted to engage different types of memory.

The Morris water maze is one of the most widely used behavioral tests for studying spatial learning and memory. In the initial phases of this task, ...

Advanced Biology

Barnes Maze: A Method For Assessing Spacial Learning and Memory in Lab Animals

Source: Ingersoll, J. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/58008/analyzing-spatial-learning-prosocial-behavior-mice-using-barnes-maze">Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms</a>. J. Vis. Exp. (2018).
This video shows how to use the Barnes Maze with a mouse. The Barnes Maze testing procedure is used by researchers to assess spatial learning and memory in laboratory animals following repeated trials.

Labirynt Barnesa: metoda oceny uczenia się przestrzennego i pamięci u zwierząt laboratoryjnych

Source: Ingersoll, J. et al. Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms. J. Vis. ...